Binding affinity-based intracellular drug detection enabled by a unimolecular cucurbit[7]uril-dye conjugate

Label-free fluorescence-based chemosensing has been increasingly brought into focus due to its simplicity and high sensitivity for intracellular monitoring of molecules. Currently used methods, such as conventional indicator displacement assays (IDAs), pose limitations related to dissociation upon dilution, random diffusion of the released indicators, and high sensitivity to interference by agents from the ambient cellular environment (e.g., salts, enzymes, and proteins). Herein we report a potentially widely applicable strategy to overcome the limitations of conventional IDAs by employing a macrocyclic cucurbit[7]uril (CB7) host covalently coupled to a nitrobenzoxadiazole (NBD) fluorescent dye (CB7-NBD conjugate). As a proof of concept, we demonstrated that the CB7-NBD unimolecular conjugate responded to various target analytes even in the complex live cell system. Moreover, the sensing system was compatible with fluorescence imaging, fluorescence-assisted cell sorting (FACS), and fluorescence spectrometry with a microplate reader. These experiments demonstrated an application of covalently bound unimolecular CB7-NBD conjugate as a sensor for detecting diverse analytes in the intracellular compartment of live cells.


Materials and Methods
Materials.All solvents were used as received from suppliers without any further purification.The concentrations of cucurbit [7]uril-tetraethylene glycol-nitrobenzoxadiazole (CB7-NBD) stock solutions were determined by fluorescence titration against a known concentration of amantadine hydrochloride by exciting the sample at 475 nm and collecting the emission intensity at 550 nm in Milli-Q water.
Synthesis.The CB7-NBD conjugate was synthesized as previously described. 1Briefly, the reporter dye NBD was attached to a TEG flexible linker, and this complex was subsequently coupled with CB7 via an Azide-Alkyne Huisgen Cycloaddition reaction.For further details regarding the synthesis, please refer to 1 .
Fluorescence spectra and binding titration experiments.Steady-state emission spectra and time-resolved emission profiles were recorded on a Jasco FP-8300 fluorescence spectrometer (Jasco Deutschland GmbH, Pfungstadt, Germany) equipped with a 450 W Xenon arc lamp, double-grating excitation, and emission monochromators.Emission spectra were corrected for source intensity (lamp and grating) and the emission spectral response (detector and grating) by standard correction curves.
All titration and kinetic experiments were carried out at 25 ºC using a water thermostatic cell holder STR-812, while the cuvettes were equipped with a stirrer allowing rapid mixing.For fluorescence-based titration experiments, PMMA cuvettes with a light path of 10 mm and dimensions of 10 × 10 mm from Brand GmbH (Wertheim, Germany) with a spectroscopic cut-off at 300 nm were utilized.
The binding titration isotherms were fitted with a direct binding model using our freely available thermosimfit software package (https://github.com/ASDSE).The estimated measurement and fitting errors of the reported log K a values is ±0.2.
Cell culture.Human hepatocellular carcinoma (HepG2) cells (HB-8065™, American Type Culture Collection, VA, USA) were kindly provided by Prof. Ute Schepers (Institute of Functional Interfaces at Karlsruhe Institute of Technology, Germany).Cells were cultured in DMEM with phenol red supplemented with 10 % v/v FBS and 1 % v/v P/S (growth medium) in a humidified atmosphere at 37 °C with 5% CO 2 .This same supplemented medium was used for all subsequent experiments unless otherwise stated.
Cell viability.The cell viability of CB7-NBD towards HepG2 cells was tested by a commerciallyavailable MTT-based assay following the manufacturer's instructions (CellTiter 96 ® , Promega GmbH, Walldorf, Germany).HepG2 cells were seeded in 96-well plates (5 × 10 4 cells/mL, 100 μL/well) and incubated overnight to let the cells attach.Then the cells were treated with various concentrations of 6.25,12.5,25,50,and 100 μM) for 72 h.After this, 10 μL of an MTT solution was added to each well and incubated at 37 °C and 5 % CO 2 for 4h.The stop solution was then added and incubated overnight at 37 °C prior to measuring absorbance at 595 nm wavelength.Cells without CB7-NBD treatment (solely cell medium) were set as the positive control, and cells exposed to cell medium with Triton X-100 at 10 % v/v were set as the negative control.
Analyte displacement assay.The HepG2 cells were incubated with growth medium supplemented with 50 μM CB7-NBD for 24 h.Next, the medium was removed, and the cells were successively washed three times with DPBS (-/-).Afterward, cell growth medium with 500 μM amantadine was added to each well and incubated for 15 min.Then, the green fluorescence was observed by a Keyence BZ-X810 fluorescence microscope at 20× magnification (λ ex = 470/40 nm, λ em = 525/50 nm) or measured by a SpectraMax iD3 microplate reader (San Jose, CA, USA).FACS analysis.The HepG2 cells were trypsinized as cell suspension and seeded into 12-well plates at a density of 5.0  10 5 cells/well.The cells were then treated with 50 μM CB7-NBD in cell growth medium for 24 h, followed by incubation with amantadine, nandrolone, and Phe-Gly at a concentration of 500 μM in cell growth medium.Then the cells were detached, washed three times with DPBS (-/-), and immediately analyzed by FACS.The mean fluorescence intensity of 1.0  10 4 cells were analyzed for each sample.
Indicator displacement measured by a microplate reader.The HepG2 cells were trypsinized as cell suspension and seeded into 96-well plates (Corning ® 96-well Half Area High Content Imaging Glass Bottom Microplate, Corning, USA) at a density of 1.5  10 4 cells/well.The cells were then cultured overnight to let the cells attach to the plate and then treated with 50 μM CB7-NBD in cell growth medium for 24 h, followed by incubation with diverse analytes (Table 1) at the concentration of 500 μM in cell growth medium.The cells were washed with DPBS (-/-) three times and immediately analyzed by a microplate reader (SpectraMax ® iD3 plate reader, CA, USA) for fluorescence intensity.

Figure S1 .
Figure S1.(A)Emission spectra of 1 μM CB7 and 1.2 μM acridine orange (AO) (λ ex = 480 nm) upon addition of amantadine (0 to 2.4 μM) in DMEM cell culture media.Please be aware that cell culture media inherently exhibits chromophoric and emissive properties.As such, potential alterations to the absorbance or emission signal of the chemosensors, induced by the addition of an analyte, may be seen only as emerging or vanishing spectral shoulders.(B) Chemical structure of AO.

Figure S2 .
Figure S2.(A) Emission spectra of 1 μM CB7 and 1.2 μM berberine chloride (BC) (λ ex = 350 nm) (non-covalent bonding) upon addition of amantadine (0 to 2.4 μM) in DMEM cell culture media.(B) Emission spectra of the covalent CB7-BC conjugate at a concentration of 1 μM (λ ex = 350 nm) upon addition of amantadine (0 to 2.4 μM) in DMEM cell culture media.Please be aware that cell culture media inherently exhibits chromophoric and emissive properties.As such, potential alterations to the absorbance or emission signal of the chemosensors, induced by the addition of an analyte, may be seen only as emerging or vanishing spectral shoulders.(C) Chemical structure of BC.

Figure S4 .
Figure S4.Chemical structures of the compounds used as analytes for the IDAs with the CB7-NBD unimolecular conjugate.

Figure S8 .
Figure S8.CB7-NBD ⊃ insulin.Plot of emission intensity at 550 nm of 1 μM CB7-NBD in DMEM media at 25 ºC, upon addition of insulin, λ ex = 475 nm.Intervals between titration steps: 250 seconds.The lines connect the data points to guide the eye and do not represent a fitting curve.

Figure S14 .
Figure S14.FACS scatter plots of HepG2 cells incubated with CB7-NBD after exposure to sole culture medium (control).The refinement of the cellular population of interest was performed by defining multiple gates or regions of interest (ROI) according to their forward and side scatter area and width parameters.The green fluorescence histogram of the control (Figure 3C, main manuscript) was calculated from the ROI depicted in the scatter plot on the right.

Figure S15 .
Figure S15.FACS scatter plots of HepG2 cells incubated with CB7-NBD after exposure to amantadine in cell culture medium.The refinement of the cellular population of interest was performed by defining multiple gates or regions of interest (ROI) according to their forward and side scatter area and width parameters.The green fluorescence histogram of the amantadine group (Figure 3C, main manuscript) was calculated from the ROI depicted in the scatter plot on the right.

Figure S16 .
Figure S16.FACS scatter plots of HepG2 cells incubated with CB7-NBD after exposure to nandrolone in cell culture medium.The refinement of the cellular population of interest was performed by defining multiple gates or regions of interest (ROI) according to their forward and side scatter area and width parameters.The green fluorescence histogram of the nandrolone group (Figure 3C, main manuscript) was calculated from the ROI depicted in the scatter plot on the right.

Figure S17 .
Figure S17.FACS scatter plots of HepG2 cells incubated with CB7-NBD after exposure to Phe-Gly in cell culture medium.The refinement of the cellular population of interest was performed by defining multiple gates or regions of interest (ROI) according to their forward and side scatter area and width parameters.The green fluorescence histogram of the Phe-Gly group (Figure 3C, main manuscript) was calculated from the ROI depicted in the scatter plot on the right.